Membrane traffic and signalling - Flashcards
Movement to late endosome – what happens to material if it has been targeted for degradation?
How does this happen?
What is the signal for sorting to the late endosome?
- If material has been targeted for degradation it will be sorted into INTRALUMENAL VESICLES giving rise to formation of the MVB
- This happens by inwards budding, then a sorting step will occur from the MVB to the late endosome
- The signal for sorting into the late endosome = UBIQUITIN (it binds to a receptor)
UBIQUITIN signal - what is it a signal for, what does it cause when added to proteins?
Ubiquitin a signal for degradation by the proteasome of soluble componants
Addition of it to proteins = reversible post translational modification
How could you test ubiquitin is the receptor signal? (show it is sufficient or necessary)
NECESSARY - add ubiquitin signal to lysines, identifying the binding site, then remove and repeat to see if ubiquitin is necessary to sort to the correct location
SUFFICIENT - add to a protein that wouldn’t normally be sorted to the lysosome and see if the signal is sufficient to target it there
MVBs are seen in the late endosome – how can these be visualised?
What determines the no vesicles within the MVB?
Can be visualised by E.M - electron microscopy
Lots of vesicles contained within the late endosome - CARGO determines the number of vesicles
Why does cargo recruit endocytic machinery and if cargo is sorted into ILVs, what does this generally mean and what happens to it?
Endocytic machinery recruited to enable proteins to be sorted to a particular place in the cell
If cargo is sorted into ILVs this generally means they have been tagged for degradation and is taken to the lysosomes to be broken down
ESCRT-Os - what process do these proteins mediate and where do they bind in order to do this?
How does this relate to clathrin?
Inwards budding to form the MVB occurs via the action of protein complex ESCRT-O (HRS + STAMP proteins.)
Hrs binds to the PI3P adaptor and recognises cargo with attached Ubiquitin (tag for degradation).
Hrs protein (part of ESCRT-O) is involved in the formation of FLAT CLATHRIN lattices (where is it functionally equivalent to an adaptor molecule)
What are the 4 cytosolic protein complexes that mediate transport from early to late endosomes?
What occurs in this sequence of proteins?
ESCRT 0: binds ubiquitnated cargo
ESCRT I: recogniton of ubiquitnated cargo, activation of ESCRTII
ESCRT II: oligomerisaton of proteins to initiate ESCRT III
ESRCT III: cargo concentration into the vesicle, deubiquitination (via de-ubiquitination proteins)
A linear progression along the complex occurs until incision and budding off is seen at the membrane
With regards to disease, why are ESCRTS important?
important for retroviral budding
Mutations give rise to cancer and neurodegneration
What provides the energy needed for the process carried out by ESCRTS?
VPS54 ATPase - required for ATP > ADP + Pi for energy needed in process of inwards budding
ESCRTS - clinical importance - how do viruses use these proteins?
How can mutations give rise to cancer?
- VIRUSES - Some viruses use ESCRT proteins to bud away from cells. They promote outward budding to HIJACK ESCRTS and allow their release from the cell.
- CANCER - ESCRTS send cargo to be degraded such as EGFs. Mutations in ESCRTS could mean this cargo isn’t degraded —»> CANCER
- NEURODEGEN - mutations in this machinery can give rise to neurodegeneration, possibly because of its link to autophagy pathway (needed to get rid of protein aggregates) – > defects in late endosome formation = disrupted autophagy = neurodegeneration
RTKs - these have multiple signalling outputs. But what do they depend on?
-CELL CONTEXT - eg type of cell, what is trafficked to where and what is required
How is RTK signalling regulated – what about other receptors (relation to endocytosis)?
Regulation of trafficking can control RTK signalling outputs - once an RTK is sorted into an MVB, receptor tail no longer accessible and signalling is turned off
–> endocytosis important for signal attenuation – material taken from cell surface and tagged for degradation therefore turning off signalling (REGULATES SIGNALLING).
Why is location of receptor key for signalling, using TGFb as an example
- TGFb is needed for growth and differentiation of cells.
- TGFb Receptor activation leads to the phosphorylation of Smad2 (downstream effector which upon phosphorylation translocates to the nucleus)
- Smad2 transcription occurs via SARA which is localised to the ENDOSOME – TGFb therefore must mediate downstream effectors by being localised to the ENDOSOME
Route of entry to cell often determines downstream signalling - explain how TGFb is an example of this
TGFb can enter cells via either -clathrin coated pits or -caveolae (endocytic structures)
- Those that enter via clathrin coated pits (where TGFb receptors are found) are delivered to early endosomes where signal transduction can occur (via Smad and SARA)
- Those that enter via caveolae (which also have TGFb receptors) are delivered to vesicles targeted to the degradative pathway
EGFrs and ligand concentration - how to concentrations of ligands determine?
WHat is the signal to switch between the pathways
Low concs EGF - uptake via clathrin mediated endocytosis —-> signalling is needed at low concs
High concs EGF – clathrin independant pathway – signals are turned off as signalling isn’t needed at high concs
Signal to switch between the pathways –> RECEPTOR UBIQUITINATION
